Duplication of the genetic matierial is performed by a multiprotein replisome machine. The replisome of E. coli, the only cellular replisome that can be reconstituted in vitro, contains DnaB helicase, a homohexamer that unwinds DNA, primase, to initiate lagging strand fragments, and DNA polymerase III (Pol III) holoenzyme that utilizes two polymerases for concurent leading and lagging strand synthesis. The holoenzyme is composed of three major components: 1) 2-3 catalyitic Pol III cores, 2) b clamps that encircle DNA for high processivity, and 3) a clamp loader that places b clamps on primed sites. The replisome is organized by t subunits of the clamp loader that bind Pol III core and DnaB. During replication of long chromosomes the replisome encounters a variety of obsticals. The replisome can not fail in passing obsticals, unlike machines that make RNA or protein where intermittent failure is not a matter of life and death. One replisome obstical is RNA polymerase, one of the tightest DNA binding proteins. We examine the detailed mechanism by which the replisome displaces a codirectional RNA polymerase and how it recruits the mRNA for synthesis. We also propose the study of head- on collisions with arrays of RNA polymerase, and to identify helicases that help the replisome resolve these collisions. Another obstical is a DNA lesion, which will halt the high fidelity Pol III. We find that translesion polymerases, Pols II and IV, take over the replisome and slow the helicase to give time for DNA repair and move the replisome past blocks. We will apply single-molecule microscopy, along with ensemble assays, to understand the mechanism of this important process. Recent studies show the holoenzyme contains 3 Pol III cores. The 3rd Pol III core may be used to bypass lesions, which we will test. Because the clamp loader is an asymmetric structure it confers asymmetry onto the Pol III cores. We will also use single-molecule methods to determine which Pol III cores function on leading and lagging strands. During lagging strand synthesis, Pol III core is sometimes signaled to release from DNA before an Okazaki fragment is complete, leaving a ssDNA gap. We will study the source of the signal, size and frequency of ssDNA gaps, and whether the 3rd Pol III core participates in this post-replication repair. Chromosome duplication is a central life process, and therefore the replisome offers an ideal target for antibacterial compounds. We have identified and solved the structure of a small compound inhibitor bound to the b clamp and propose to expand this project, performing chemistry in collaboration with the Broad Institute. We also propose to use Rockefeller University's high throughput screening facility to identify inhibitors to other replisome components. Cellular replisomes from all three domains of life share similar strategies to the E. coli replisome. Hence, the lessons learned from this proposal should generalize. Finally, replication gone awry forms the basis for diseased states, including cancer. Understanding the basic processes of chromosome duplication may aid development of drugs for therapy of diseased states that involve the replication process.